The scope of chemical reactions which rely on auxiliary species for promoting the reaction and/or directing its course is large and varied. The term "auxiliary species" is used herein to denote ions, molecules or complexes which interact with a substrate to promote its conversion, but do not become chemically bound to the substrate in the final product. Such reactions include those occurring both in and outside of the biological context.
One subset of these reactions are those in the biological context, where in the typical case the auxiliary species normally functions in coaction with a protein, which may be an enzyme or some other macromolecular species. The auxiliary species in such reactions is a cofactor or non-protein component which, in combination with the enzyme, enters into or promotes the reaction mechanism in any of a variety of ways. Cofactors of this type vary widely in chemical composition, including both organic and inorganic species, and including species which are loosely associated with the enzyme as well as prosthetic groups tightly associated with the enzyme. Other subsets of the reactions are reactions which do not occur as part of a biological process, with auxiliary species which are not biologically derived.
It has now been discovered that reactions involving the use of auxiliary species can be promoted by antibodies whose antigen binding sites have a conformation favorable to the reaction, either by increasing the reaction rate, improving the selectivity, or affecting some other parameter in a manner favorable to the progress of the reaction toward the desired product. The terms "antibody" and "antibodies" are used herein to include both whole antibodies and antibody fragments.
For those reactions which normally occur in the biological context, the antibodies in accordance with this invention serve as a replacement for enzymes, and function in combination with the cofactors to produce a result generally equivalent to that achievable with the enzymes. The antibodies thus function in a reaction-promoting manner analogous to the enzymes. The antibodies, like the enzymes which they replace, achieve this effect in a variety of ways, depending on the particular chemical reaction for which they are elicited and used. They may, for example, restrict the rotational and translational motions of the reacting species and/or cofactor, stabilize conformations of the reacting species and/or cofactor in a manner favorable to the reaction, stabilize transition state complexes of the reacting species and the cofactor, vary the electronic configuration of the reacting species and/or cofactor in a manner favorable to the reaction, or any other means of promoting the reaction.
In reactions normally occurring in contexts other than biological, the antibodies in accordance with this invention serve an analogous function, increasing the reaction rate, improving selectivity, or generally promoting the formation of products which might not otherwise be formed.
Reactions to which the present invention is applicable include both intramolecular and intermolecular reactions, including rearrangements, cyclizations, condensations, hydrolytic reactions, additions, eliminations, isomerizations, reductions and oxidations. Accordingly, substrates to which the present invention may be applied range in size from relatively small molecules of ten atoms or less to macromolecules such as proteins, hormones, polysaccharides and polynucleotides. Examples of such reactions are selective peptide and oligosaccharide hydrolysis, thiol oxidation, stereospecific alcohol oxidation, stereospecific ketone reduction, transamination reactions of keto acids to amino acids, glycosylation reactions, transacylation reactions of peptide esters, and phosphodiester hydrolysis.
Auxiliary species to which the present invention is applicable may likewise vary widely in size, ranging from monatomic metallic ions through small molecules of ten atoms or less to larger molecules with complex three-dimensional structures. As indicated above, the term "auxiliary species" is used herein to denote any species which, when used in accordance with the present invention, will function in coaction with an appropriate antigen binding site to promote the progress of a chemical reaction. The term "cofactor" will also be used in a general sense, to include all such auxiliary species.
Such species are not a part of the starting material or product, and may or may not undergo a change as the result of the reaction. These species, if used in conjunction with an enzyme rather than an antibody as in the present invention, would otherwise be termed "coenzymes," "activators," or "prosthetic groups," depending upon their structure and the means by which they are associated with the enzyme. Examples of such auxiliary species are nicotinamide coenzymes such as nicotinamide adenine dinucleotide (NAD) or its reduced form (NADH) or nicotinamide adenine dinucleotide phosphate (NADP), flavins, cobamides, cobalamins, ascorbic acid, ferredoxin, thiamine pyrophosphate, pyridoxal-based compounds such as pyridoxal phosphate, pyridoxol and pyridoxamine, tetrahydrofolic acid, biotin, S-adenosylmethionine, coenzyme A, purine phosphates, pyrimidine phosphates, glutathione, metal ions such as Cu, V, Fe, Zn and Co, and complexes of metal ions with ligands such as bipyridyl, phenanthroline, EDTA and porphyrins.
In the practice of the present invention, the auxiliary species are not covalently bound to the antibodies. The antibodies are thus formed separately from the auxiliary species, with binding sites which include regions which are complementary to the species in a manner which promotes the reaction by restricting the species to conformations, orientations, or electronic or steric characters which promote the progress of the reactions. In certain cases, the antigen binding sites are complementary to a transition state complex of the auxiliary species and the substrate, or any other high energy complex along the reaction coordinate leading to the reaction product.
Methods of eliciting antibodies for a particular reaction involve the use of a hapten designed to approximate the reactive moiety or moieties, including the auxiliary species itself as well as a substrate binding site, in the rotational, translational and electronic conformations needed for the reaction to proceed. In certain cases, the hapten will be a stable analogue of a transition state favorable to the reaction, the analogue being one in which an unstable portion of the structure has been replaced with a stable group of similar size, shape, orientation and electronic configuration. In further cases, the hapten will be a stable analogue that mimics an unstable intermediate on the reaction pathway. The analogue in such cases will be one which can be synthesized and isolated in high purity, unlike the intermediate itself. In still further cases, the hapten will be a hybrid or conjugated structure containing moieties analogous in steric and electronic conformation to the auxiliary species and the substrate. The arrangement of these moieties in the hybrid structure will closely approximate the actual auxiliary species and substrate in the relative orientation and spacing needed for the reaction to proceed. The present invention extends to still further types of haptens and the microenvironments which they create in the antigen binding sites, as will become apparent from the description which follows.
The present invention further entails in certain cases the discovery of stable haptens bearing the characteristics described above, and their use in eliciting the appropriate antibodies. In some of the reactions disclosed herein, the haptens are specifically designed to elicit antibodies which, although enhancing the progress of the reaction and the formation of the desired product, avoid product inhibition of the antibody binding capacity. This effect is also achieved in some cases by appropriate selection of the reactive species among alternative systems all of which are capable of forming the desired product. In some cases, antibodies will be generated to a specific isomer of the reacting species such that the products of the reaction have a stereocenter.
The use of the haptens in generating the antibodies follows conventional procedures involving host immunization. The haptens are generally coupled to carrier molecules which render them immunogenic, the coupling achieved through conventional linking groups or spacers. Further advantages in both quantity and specificity may be obtained by the use of monoclonal antibodies.